Effort to produce renewable alternative sources of transportation fuels from biomass have resulted in considerable progress in the conversion of hardwood and agricultural waste into ethanol. Added yields could be expected if the pentosan hemicellulose (five carbon sugar polymers), in addition to hexoses and cellulose, could be effectively fermented to ethanol.
Softwood Prehydrolysates
However, except for sulfite waste liquor, reports of the conversion of softwood materials to ethanol have been limited. Furthermore, the hemicellulose for most softwoods studied is primarily hexosan, composed mainly of mannose with smaller amounts of glucose and galactose, as well as some pentoses. Traditional Saccharomyces cerevisiae yeast cultures ferment these hexoses very well, and would be expected to produce high ethanol yields if they could tolerate low concentrations of countless toxins present in dilute acid prehydrolysates generated from softwoods. Typically, these prehydrolysates are generated to produce monomeric sugars from carbohydrate polymers and/or to improve enzymatic digestibility of cellulose in forest waste, municipal solid waste (MSW), or agricultural waste. This biomass is usually converted into a prehydrolysate slurry by soaking the biomass in dilute acid (0.1% to 4%), draining, and then steaming it at about 170xc2x0 to 215xc2x0 C. for 30 to 360 seconds.
Prehydrolysates from softwoods are believed to be more toxic than those from agricultural wastes or hardwood biomass sources, because softwoods usually contain more extractives and often more bark than do hardwoods. Consequently, if a process is found to ferment softwood prehydrolysate, fermentation of other prehydrolysates should directly follow. Potential toxic substances include biomass components themselves, particularly extractives such as terpenes, aldehydes, and polyhydroxy aromatics. Other sources of toxins are prehydrolysis products and degradation products including acetic acid from acetylated sugars, furfural, and hydroxy-methyl furfural, the initial degradation products from pentose and hexose sugars, respectively, and oligomers formed by reaction of the furfurals with sugars. Degradation of coniferous lignin yields complex guaiacyl propyl units. Corrosion products from equipment also can be toxic, or the metallic ions can behave as catalysts to produce additional products.
Fortunately most of the toxins in well-prepared prehydrolysate are present at less than one g/L, and only a very few, such as furfural, are present at a few g/L. However, over time, yeast can adapt themselves to tolerate many of these substances in the presence of glucose sugarxe2x80x94but the adaptation in the presence of these toxins prevent or greatly reduce growth and ethanol production.
Yeast Bioreactions and Fermentation
It is generally well known that Saccharomyces cerevisiae yeast aerobically oxidizes low concentrations of sugars in aqueous solutions to produce yeast cell mass, carbon dioxide and water, in accordance with the following equation:
1) C6H12O6 (xcx9c100 g dry wt.)+6O2xe2x86x926 CO2+6H2O+Cell Mass (xcx9c50 g dry wt.)
Under these conditions little or no alcohol is produced. This is how bakers yeast is produced. However, at higher concentrations of sugar, even in the presence of much air, the sugar shuts down the oxidative metabolism of the yeast (the Crabtree effect), and the yeast then ferments the sugars to ethyl alcohol (ethanol), one-third the amount of carbon dioxide, much less cell mass, and no measurable amount of water in accordance with the equation:
2) C6H12O6 (xcx9c100 g dry wt.)xe2x86x922 CH3 CH2 OH (ethanol, xcx9c51 g)+2CO2+Cell Mass (xcx9c5 g dry wt.)
This is a forced xe2x80x9cfermentation,xe2x80x9d with air or without air, because of the Crabtree effect. In the correct sense, fermentation is a term for conversion of sugar to ethanol. Unfortunately, this term has been loosely used for any type of microbial metabolism, even bio-oxidation, by any organism. The loose use of the term fermentation has lead to considerable confusion.
For example, in much of the prior art, even the oxidative conversion of sugars primarily to bakers yeast cell mass is referred to as fermentation.
Prior Art
U.S. Pat. No. 5,693,526 disclose novel strains of yeast S. cerevisiae for ethanol production using a number of different hybridization methods for obtaining improved flocculation, improved growth on 40% to 50% molasses (high sugar concentration), and growing in the presence of 7% to 12% ethanol. In this reference, there is no disclosure of hydrolysate, little or nothing on aeration, and little or nothing on low nutrient or on simultaneous aeration, low nutrient, and hydrolysate adaptation.
A process for making yeast tolerant to high pressure in which yeast are transformed with foreign DNA to encode for two enzymes, superoxide dismutase and catalase is disclosed in U.S. Pat. No. 5,674,721. This patent tests the transformed cells to show that they are twice as heat tolerant as the original yeast cells by heating them in the air at about 50xc2x0 C. The patent does not disclose hydrolysate, evidences little or no discussion on aeration during growth or fermentation, evidences no appreciation for low nutrient or simultaneous aeration, and hydrolysate adaptation.
U.S. Pat. No. 4,567,145 disclose used respiration deficient yeast, and makes no reference to respiration enhanced yeast. This patent does not claim tolerance to hydrolysate nor adaptation to low nutrients. Further, there is no reference in this patent to simultaneous aeration, low nutrient, and hydrolysate adaptation.
Processes for screening aerobic yeast produced from hybridization or mutation using two tests for bread-making which do not use gas-release measurement, such as more growth on maltose, or more growth on sour (acid) dough are disclosed in U.S. Pat. Nos. 4,396,632; 4,318,929 and 4,318,930. These patents generate their yeast by hybridization and mutation, and test these yeast to determine whether they have improved in their performance. These patents do not generate their new strains naturally by simultaneous acclamation under severe constraints over time. Further, the processes in these patents are fully aerobic yeast metabolism, for making bakers yeast. These processes are not fermentation producing ethanol.
U.S. Pat. No. 4,477,569 disclose pentose fermentation by yeast Pachysolen tannophilus (not S. cerevisiae) with air in the first stage and with improved yield with recycle. The pathway in this process is different and this pathway is not known to exist in S. cerevisiae. This patent uses a very rich, very expensive medium including yeast nitrogen base, yeast extract, and casamino acids along with its sugars. Further, this patent states that air is not needed with cell recycling and also uses the mutagen, ethyl methanesulfonate. The patent does not use hydrolysate and does not adapt its cells to tolerate the toxic hydrolysate at very low, inexpensive nutrients simultaneously.
An aerobic fermentation is disclosed for production of high-density yeast cell mass, but not ethanol, in U.S. Pat. No. 4,414,329. The high cell densities are produced in a continuous stirred tank bioreactor by continually feeding mineral salts with the carbon source feed, to eventually achieve cells with high mineral content.
U.S. Pat. No. 3,384,553 disclose a method for aerobic fermentation in which the dissolved oxygen concentration in liquid medium for cultivation of microbes is controlled by the rate of addition of medium to the culture. This patent does not disclose the actual production of ethanol by true fermentation:
xe2x80x9cYeast Adaptation on Softwood Hydrolysatexe2x80x9d, in Applied Biochemistry and Biotechnology 70-72, 137-148 (1998) disclose that the highest equivalent total solids (ETS) fermentable hydrolysate concentration is up to 17%, at which concentration we learned that the culture stalled out. In the publication, the concentration of the culture was dying off faster than it was growing, and anything higher than 17% could not be sustained at that time. Therefore, there was a need to diagnose the reason for the stalling out and to over come it by forcing the yeast mitochondria to function.
Accordingly, a need existed to obtain yeast functioning mitochondria at high sugar level concentrations, which normally repressed yeast mitochondria.
There is also a need in the art of utilizing yeast to ferment ethanol from sugars to provide new strains of yeast able to use oxygen for growth while fermenting xe2x80x9cCrabtree negative,xe2x80x9d normally toxic concentrations of hexose containing wood prehydrolysates to ethanol.
There is further need in the art of fermenting sugars to ethanol from toxic levels of prehydrolysates sugars, to also produce more cell mass, that is not possible in typical xe2x80x9cCrabtree positivexe2x80x9d yeast fermentation of sugars.
A yet further need in the art of producing ethanol from fermentation of sugars in the normally toxic softwood prehydrolysate environment is to provide new strains of yeast that are not contaminated by any wild organism.
One object of the present invention is to provide new yeast strains of Pichia stipitis that are able to use oxygen for growth while fermenting normally toxic concentrations of xylose containing wood prehydrolysates to ethanol.
Another object of the present invention is to provide new yeast strains of Pichia stipitis capable of not only producing ethanol from toxic levels of prehydrolysate sugars, but also producing more cell mass.
A further object of the present invention is to provide new strains of the yeast Pichia stipitis capable of producing ethanol from toxic levels of prehydrolysate sugars, while producing more cell mass, by controlling aeration and nitrogen-source levels.
A still further object of the present invention is to provide new strains of the yeast Pichia stipitis able to use oxygen for growth while fermenting normally toxic concentrations of xylose as well as hexose containing wood prehydrolysates to ethanol, wherein the new yeast strains have the ability of maintaining a growth rate in excess of the death rate in toxic fermentation broth.
A yet further object of the present invention is to provide a new yeast strain of Pichia stipitis, designated NPw9 that is cultured in normally toxic wood prehydrolysates, that has never been contaminated by any wild organism.
A still further object of the invention is to provide a new yeast strain of Pichia stipitis Npw9 capable of fermenting equivalent total solids (ETS) hydrolysates at concentrations in excess of 17% without incurring the culture stall-out experienced at this level of equivalent total solids, by achieving functioning mitochondria at higher than 17% hydrolysate concentrations by reducing the nutrient concentration, while increasing aeration, and simultaneously increasing the hydrolysate concentration.
A further object of the present invention is to provide new yeast strains of Pichia stipitis NPw9, capable of using oxygen for growth while fermenting xylose in wood prehydrolysates to ethanol that are not only cost-effective, reducing the need for sterilization, but very robust and ideal for large-scale commercial production.
A further object yet still of the present invention is to provide a new yeast strain of Pichia stipitis of designation NPw9 that is cultured in normally toxic wood prehydrolysates, that has never been contaminated by any wild organism.